Combining ability, heterosis and performance of grain yield and content of Fe, Zn and protein in bread wheat under normal and late sowing conditions

Wheat (Triticum aestivum L.) is inherently low in protein content, Zn and Fe. Boost yield gains have unwittingly reduced grain Zn and Fe, which has had negative impacts on human health. The aim of this study was to understand the inheritance of grain yield per plant and grain Fe, Zn, and protein concentrations in bread wheat (Triticum aestivum L.) under normal and late sown conditions. Half diallel crosses were performed using 10 parents. The crosses and parents were evaluated in replicated trials for the two conditions, to assess the possibility of exploiting heterosis to improve micronutrient contents. The per se performance, heterosis, combining ability, and genetic components were estimated for different characters in both environments. The results revealed that hybrid GW 451 × GW 173 exhibited better parent heterosis (BPH) and standard heterotic effects (SH) in all environments. In both sowing conditions, the general combining ability (GCA) effects of poor × poor parents also showed high specific combining ability (SCA) effects of hybrids for both the micronutrients and protein contents. However, σ2A/σ2D greater than unity confirmed the preponderance of additive gene action for protein content, and GW 173 was identified as a good general combiner for these characteristics under both environments. SCA had positive significant (P < 0.001) correlations with BPH, SH1, SH2, and the phenotype for yield component traits and grain protein, Fe, and Zn concentrations in both conditions. A supplementary approach for biofortifying wheat grainis required to prevent malnutrition.


INTRODUCTION
Wheat (Triticum aestivum L.) is a crucial staple food crop worldwide, and has been consumed since ancient times by human beings (Giraldo et al., 2019). Worldwide, wheat is cultivated on more than 17% of all cultivable land, occupying approximately 32% of the total land under cereal cultivation. It is consumed by nearly 40% of the global population, and fulfills 21% of the daily protein requirements of more than 4.5 billion people in developing countries (Giraldo et al., 2019). Micronutrient malnutrition is a serious public health concern in several parts of the world, especially underdeveloped countries (Ragi et al., 2021). Hidden hunger, or micronutrient deficiency is one of major reasons for anemia. Currently, 60% and 30% population is deficient in Fe and Zn, respectively (Ragi et al., 2021). In India, 80% of pregnant women, 52% of non-pregnant women, and 74% of children aged approximately 6-35 months suffer from induced anemia-Fe deficiency (IDA) (Sharma et al., 2021). Biofortification can be a useful tool to fight this "hidden hunger," playing a crucial role in the improvement of micronutrient contents in the diet, which will benefit billions of people (Bouis & Saltzman, 2017). Micronutrient deficiencies in humans could be altered through biofortification, mineral supplementation, and food diversification and fortification (Gupta, Hossain & Muthusamy, 2015). Biofortification is the action of increasing the content of micronutrients in the edible parts of staple food crops to ensure better human nutrition (Prasanna et al., 2020). However, the root cause of micronutrient malnutrition is staple crop-based diets that generally do not furnish the required amounts of heavy assurance and micronutrients (Wakeel et al., 2018). The use of biofortified cultivars is needed to alleviate micronutrient malnourishment in large populations, especially in Africa (Sofield et al., 1977). Biofortification strategies cover breeding in wheat, genetic control, and utilization of mineral fertilizers, and have broad potential to address micronutrient malnourishment (Garg et al., 2018).
Different sowing dates are primarily connected to the effects of temperature and day duration (Chaudhari, 2022). Low wheat yields in central India are caused by the short winter season and high temperatures throughout the growing season. Here, the main effects of high temperature are decreased grain weight and reduced grain filling duration (Wardlaw et al., 1989;Mutwali et al., 2015). Escape mechanism is functional in late sown wheat to reduce the effects of high temperature during grain filling while early sown wheat use escape mechanism to cope with problem of high temperature during germination and vegetative growth. Consequently, recognition of the best wheat genotypes appropriate for particular sowing conditions is of considerable significance to attain higher yields and micronutrient uptake. Understanding associations between grain yield, protein, and micronutrient content under normal and late sown conditions will be helpful for heterosis breeding/hybrid wheat technology. Micronutrient supplementation provides a balanced nutritional diet for people who suffer from deficiencies due to dependence on a wheat grain diet for their Fe and Zn intake. Therefore, to tackle micronutrient insufficiency among wheat eaters, attempts must be made to increase micronutrients, especially grain Fe and Zn content accompanied by supportable crop grain yield. The objective of this study was to evaluate the per se performance, heterosis, and combining ability of wheat parental lines and the hybrids produced from them in terms of grain Fe and Zn content under normal and late seeded conditions. These results directly affect the efficacy of producing high-yielding hybrids with high levels of Fe and Zn densities under different environment conditions. genetic variance ((σ 2 A/σ 2 D) 0.5) were estimated. These genetic components of variance were calculated from the ANOVA table (Griffing, 1956).

ANOVA performance
In Table 1, the performance of hybrid and parental lines is presented. The mean squares of genotypic effects for the traits under study were significant at P < 0.01 in both sowing conditions.
In Table 1, the outlined diallel mating design is analyzed using ANOVA. The ANOVA for combining ability in E 1 and E 2 environments revealed that mean squares of parents and hybrids were significant (P < 0.01) for all characters.
The estimates of σ 2 GCA and σ 2 SCA revealed that additive and non-additive gene effects were involved in inheritance of GYP and Zn content in E 1 . Additionally, the estimates of σ 2 SCA were significant for all characters in both environments, which suggested the importance of only the non-additive gene effects for inheritance of these characters.
The value of the average degree of dominance was less than one for the characters GYP in E 1 , protein content in E 2 and zinc content in E 1 . Partial dominance behavior was revealed by the interaction of alleles for these characters. For the Fe content in E 1 and E 2 and zinc content in E 2 , the value of the average degree of dominance was close to zero or zero, revealing an absence of dominance for both characters.

Hybrids and parental lines performance
The per se performance of parental lines, hybrids, and checks are presented in Table 2.
In Table 2, coefficient of variation (CV) <10% for the calculated traits indicated acceptable and efficacious experiment results. The GYP in hybrids ranged from 10.0 g (H45) to 32.9 g (H3) in E 1 and from 9.4 g (H27) to 35.4 g (H24) in E 2 . The standard check HD 2932 had the highest GYP compared with MACS 6222 in both environments. The PC in hybrids ranged from 9.9% to 11.7% in E 1 and 10.6% to 12.4% in E 2 . In case of standard checks, MASC 6222 had a higher protein content compared to HD 2932 in both environments. The

Heterosis performance
Two categories of heterosis, standard heterosis (SH) and better parent heterosis (BPH), exhibited significant variation under both sowing environments (Tables 3 and 4). In this study, several crosses indicated noticeable heterotic responses over better parent (heterobeltosis) and standard check varieties (standard heterosis). These were MACS 6222 for normal sown and HD 2932 for late sown.
However, the higher percentage of hybrids showed a more positive BPH under late than normal conditions for PC. One F 1 progeny, H30, under normal conditions, and eight F 1 progenies under late conditions showed significant positive BPH. In particular, under the late condition there were higher significant positive crosses for SH than under the normal condition.
For Fe content, H5, H9, and H36 had consistently significant positive heterosis for BPH under both conditions. H5, H7, H8, H28, H30, H32, and H36 were significantly positive for SH under both checks and environments. Interestingly, H5 had consistently significant positive BPH and SH estimates for Fe content under both environments, and GYP under normal conditions (SH).
For Zn content, H45 had consistently significant positive BPH under both environments and significant positive SH over both checks, except SH 1 in E 1. In addition, H36 had the highest significant positive estimates of SH for Zn content under normal Chaudhari et al. (2023), PeerJ, DOI 10.7717/peerj.14971 5/21 conditions. Nine F 1 progenies for normal and late conditions exhibited significant positive SH.

GCA effect of parents
No parent simultaneously had positive GCA effects for all the studied characters under both environments (Table 5). For GYP and PC, P1 had significant positive GCA effect

SCA effect of hybrids
As shown in    , and significant positive effects in both environments for Fe content. In addition, the variance caused by SCA (σ 2 SCA ) was greater than GCA (σ 2 GCA ) for all studied environments (Table 1).

Correlations among per se performance, heterosis, and combining ability
The interconnection among per se performance, heterosis, and combining ability effects yield attributes, PC, and Fe and Zn content under both environments are shown in Table S3. SCA had no significant correlation with GCA effects, except for Zn content under the late condition. BPH had a significant relationship with GYP (r = 0.41) under normal conditions. When Fe and Zn contents were significantly correlated under the late condition. For SH1, SH2, and phenotype, significant relationships were observed for all traits under both environments, except Fe under the normal condition. SCA had a positive significant (P < 0.001) correlation with BPH, SH1, SH2, and phenotype for yield component traits and grain protein, Fe, and Zn concentrations under both environments. GSCA had a significantly (P < 0.01) positive association with the GCA effect. On the

DISCUSSION
Approximately two billion people, nearly one third of the global population, have been estimated to be deficient in one or more mineral components (Huseynova & Rustamova, 2010). Biofortification is one of the most effective approaches to alleviate malnutrition (Global Nutrition Report, 2017, https://www.globalnutritionreport.org). Besides the major required elements, micronutrients are also involved in regulating many vital metabolic functions (Heck et al., 2020). Biofortification is an approved strategy to fight micronutrient deficiency in large populations, particularly for those living in developing countries.
To make it more effectual, accessible, and manageable for people, well planned, applied, observed, and appraised biofortification programs are needed to produce economical and socially allowable biofortified food crops (Bouis & Saltzman, 2017;Bouis & Welch, 2010;Bouis, Saltzman & Birol, 2019). Significant advancement has been attained in developing high Fe and Zn wheat varieties for cultivation under normal and late conditions. Biofortified varieties, such as HD 3298, WB 02, HPBW 01, Pusa Tejas (HI 8759) Pusa Ujala (HI 1605) HD 3171, HI 8777, MACS 4028, PBW 752, PBW 757, Karan Vandana (DBW 187), DBW 173, UAS 375, DDW 47, PBW 771, HI 8802, HI 8805, HD 3249, MACS 4058, HI 1633, DBW 303, andDDW 48 with high PC, Fe, and Zn content have been released for commercial farming in different countries (Yadava et al., 2020). In wheat, various studies on PC, Fe, and Zn discuss biofortification. Some progress in grain yield advancement and micronutrient improvement has been made under both environments discussed in the present study. In this study, a total of 45 hybrids were developed by following the half diallel mating design by using 10 parental genotypes under normal and late environments. The hybrids, parental genotypes and two standard checks, MACS 6222-TS and HD 2932-LS, were estimated for GYP and protein, Fe, and Zn content under normal and late sown conditions. The parents, P9 and P5, were good general combiners for all the studied characters under both environments. For Fe and Zn traits, hybrid H14 was best under normal sowing and H37 for late sowing. In our study, some crosses were better for GYP than their parents and check varieties, and expressed the distinct particular cross yield under both environments. The hybrids exhibited a smaller mean value for protein content compared to the parents. In the present study, several hybrids manifested noticeable heterotic response over the better parent (heterobeltosis) and standard check varieties (standard heterosis). For example, MACS 6222 was best for normal sown and HD 2932 for late sown. Between the two groups of heterosis, SH was more appropriate than BPH. SH illustrated the acceptance yield edge of hybrid varieties over the best checks. Under both environments, hybrids exhibited negative heterosis for all the calculated characters.
For PC, the extent of HB was low to high in the negative direction and low in the positive direction. However, the magnitude of SH was moderate in the positive and low to high in the negative direction. The findings agreed with the results of previous studies (Kumar, 2012;Desale & Mehta, 2013;Chaudhari & Patel, 2014a;Ekhlaque, Kamar & Jaiswal, 2016) for HB. The findings for SH were in accordance with those of Kumar et al. (2018) and Chaudhari et al. (2017). For Fe content, the magnitude of HB was moderate in the negative and low in the positive direction. The magnitude of SH was moderate in both directions. The results contradicted the finding of Younas et al. (2019). For Zn content, the magnitude of HB and SH was high in the negative and low in positive direction.
The differential parental lines using GCA illustrated the average performance of the parental line in an array of hybrid crosses. In this study, the effects of GCA in parental lines for protein content varied with the environment. The P1 had the highest significant positive GCA effect in E 2 (0.20). Another good general component was P7 in E 1 (0.29). P10 was considered a poor general component because it possessed a significant negative GCA effect under all environments. Under both environments, the effects of GCA were at odds with the grain Fe content of parents, excluding P7. Under E 1 , the effects of GCA for Fe and Zn contents were contradictory. Under E 1 and E 2 , grain yield and Fe and Zn content showed an anti-positive correlation and GCA effect such that these traits can be considered to improve parental substances. Hence, these types of parents could be used in selection of desirable types to improve combination breeding.
This study revealed a high degree of correlation between the performance of parents and their GCA effects for most of the characters studied, and whether the desirable per se performance could be used for the hybridization program to select parents.
The success in crop improvement lies in isolating superior gene combinations in the genotypes with high combing ability. The genotypes with good general combining ability can be further exploited in developing a new variety.
Non-additive gene action is linked with SCA, followed by dominance over dominance and epistatic effects (Younas et al., 2019;Sprague & Tatum, 1942;Saleem, Mirza & Haq, 2010;Chaudhari et al., 2022). The findings also indicated the involvement of both additive and non-additive gene action in the inheritance of yield and related traits in wheat . Analysis of the crosses revealed that no special combination prevailed in significant positive SCA values for all the estimated characters concurrently. This result is in accordance with those from previous studies on rice (Griffing, 1956;Gramaje et al., 2020), maize (Tiwari et al., 2011), and bread wheat (Kahriman et al., 2016). However, the crosses H3, H2, and H6 (significant (P < 0.01) for GYP) and H43 (significant (P < 0.01) for grain Fe and Zn content) showed positive SCA values for all the traits in normal conditions. Oppositely, late condition-H3 (significant for GYP, PC, and grain Fe content) and H26 for grain Fe and Zn content appeared to have positive SCA. These results were in accordance with previous studies on rice (Fellahi et al., 2013), pearl millet (Velu et al., 2011b;Govindaraj et al., 2013), and maize (Long, Bänziger & Smith, 2004;Chen et al., 2007) suggesting that the grain micronutrient contents are mainly under additive genetic action.
Significant SCA effect for any character may not have significant GCA effects for parentage cross combination (Chaudhari & Patel, 2014b). For example, parents of H15 hybrids had significant negative GCA effects, and themselves had a significant positive SCA effect for yield characters in E 1 . Here, no significant SCA effects were determined in all hybrids for estimated characters. It is recommended that these characters be less than the maximum mean of parents.
In the reverse case, parents with significant GCA effects did result in the best crosses with significant SCA like parents of H2, H3, and H26 which showed positive GCA effects, and crosses were observed with positive SCA effects for GYP in normal conditions. This remark could be assigned to unlike combo of dominant and recessive genes from particular parents; it far justifies the non-additive gene action (additive × dominance, dominance × dominance, and epistatic interactions). The cross H3 was observed with the highest significant positive SCA effect for GYP in normal conditions; GYP, PC, and grain Fe content in late conditions. For grain Fe and Zn content, the hybrid H43 had a significant positive SCA effect in both environments. Generally, hybrids with high SCA effects are recommended for heterosis breeding. Thus, these hybrids could be exploited in the breeding program to select useful segregants in succeeding generations and consequently, it would be valuable to utilize them to enhance grain yield and micronutrients. Therefore, the GCA effects of parents are insufficient to anticipate the SCA effects of respective hybrids. Conversely, a significant association between SCA effects and heterosis was noticed for all studied characters in both environments. In common, heterosis breeding targeted for particular traits based on the GCA effects of parents can be taken as the best cross combinations.
These hybrids involved at least one good general combiner parent and would produce transgressive segregants. Although, for full exploitation of existing genetic variance in these hybrids, intermating of best plants in early segregating generations would be useful to assemble the best populace of parents with high micronutrients and grain yield. However, hybrids combo with high SCA estimates can be exploited in heterosis breeding. Additive gene action act for GCA, and non-additive gene action compute SCA.
In the late condition, a positive correlation between grain Fe and Zn content was noticed in parents and the hybrids. Similar associations among these grain micronutrient content have been noted formerly in rice (Stangoulis et al., 2007;Anandan et al., 2011), maize (Arnold, Bauman & Aycock, 1977), wheat (Garvin, Welch & Finley, 2006;Velu et al., 2011a), sorghum (Kumar et al., 2010;, pearl millet (Govindaraj et al., 2013;Velu et al., 2008;Rai, Govindaraj & Rao, 2012;Kanatti et al., 2014), and finger millet (Upadhyaya et al., 2011). To the best of our knowledge, this is the first report evaluating parental wheat lines and hybrids under normal and late sown conditions for grain protein and micronutrient content.

CONCLUSION
This study elucidated variation in GYP and protein, Fe, and Zn content under normal and late sown conditions. Previously, protein and micronutrient contents have rarely been estimated under normal and late sown conditions. The present findings indicate that the hybrids exhibited higher mean micronutrient and protein contents under late sown condition than normal. The study paved the way for further improvement of GYP and protein, Fe, and Zn contents under late sown conditions. However, it will be necessary to introduce high Fe and Zn densities into both parental lines in order to breed hybrids, and the use of genomics techniques may greatly speed up this procedure. Parental lines of potential hybrids with high general combining ability (GCA) could be successfully selected based on their performance, increasing the breeding efficiency, according to a highly significant and positive correlation between their performance and their GCA for both Fe and Zn densities. Further research is necessary because of the direct impact these findings have on the effectiveness of breeding high-yielding hybrids with high levels of Fe and Zn densities. Inbred lines show no correlation between Fe and Zn densities and grain yield, but hybrids show a significant negative (though low) correlation.